Mitochondrial Genetics Research Group
Research Group Head
The overall aim of the Mitochondrial Genetics research group’s program is to understand the interactions between the nuclear and mitochondrial genomes and how these processes regulate cellular function. We undertake these studies using oocytes, preimplantation embryos, embryonic and adult stem cells, somatic cells, and tumour-initiating cells. We use a number of oocyte, embryo and cell manipulation techniques, next generation sequencing technologies and bioinformatics. Our work is divided into three areas, which we will continue to interrogate over the next five years.
Research Focus One: Understanding the role of mitochondrial DNA (mtDNA) in oocyte quality and development outcomes.
We undertake this area of research using porcine and bovine oocytes and generate embryos and offspring through in vitro fertilisation (IVF), nuclear transfer (NT) cytoplasmic transfer (CT), and mitochondrial supplementation (mICSI). This enables us to determine how the nuclear and mtDNA content of an oocyte affects fertilisation and developmental outcome; and how mtDNA segregation, transmission and replication are normally regulated, as is the case following IVF, and perturbed as these mechanisms are violated by NT and CT.
We are currently funded by the NHMRC (Development Grant) to generate mini-pig models using a new assisted reproductive technology that we have been developing, which involves supplementing oocytes deficient in mtDNA with genetically identical populations of mtDNA. This technology is known as mICSI, and our published data suggest that mICSI enhances embryo quality and overcomes the predisposition of poor quality oocytes to give rise to diabetes and obesity. We have recently demonstrated the mechanism associated with this process. The grant will determine whether supplementing eggs at the time of fertilisation with genetically identical mtDNA is safe practice.
We have also previously generated a derivation of another assisted reproductive technology, namely cloning or somatic cell nuclear transfer (SCNT), which ensures cloned embryos inherit their mtDNA from the population present in the oocyte only, as is the case following natural conception, and not from the somatic cell as well. This technology, known as mito-SCNT, improves embryo quality and developmental outcomes and overcomes the problem of embryos and offspring inheriting two populations of mtDNA.
Research Focus Two: Defining the role of the mtDNA set point in early development and cancer.
In recent years, we have discovered the ‘mtDNA set point’, which is a key developmental milestone that naïve (pluripotent) cells must acquire in order to initiate and complete their differentiation into mature cell types. The mtDNA set point is characterised by cells expressing pluripotent markers and having very low mtDNA copy number, which are both regulated by the epigenetic status of the nuclear genome. Furthermore, we have shown that key nuclear-encoded mtDNA replication factors are DNA methylated in a tissue specific manner that accounts for cell-specific mtDNA copy number, which requires the establishment of the mtDNA set point early during development. Our studies have also shown that failure to establish the mtDNA set point results in developmental failure and is a key characteristic of tumour-initiating cells. In addition, by depleting tumour-initiating cells of their mtDNA, we have shown that the incidence of tumour formation is significantly reduced (glioblastoma mutiforme and osteosarcoma) or prevented (multiple myeloma), as these cells re-establish the mtDNA set point and undergo differentiation and, therefore, do not form tumours.
We use and derive embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and tumour-initiating cell lines and tumours to perform these studies. These models allow us to focus on molecular mechanisms and to identify the specific windows during differentiation/development when these events manifest and then to confirm and characterise these events through, for example, targeted gene knockdown and overexpression.
Research Focus Three: Determining how mtDNA haplotypes influence phenotype.
Recent outcomes from our research program have shown that mtDNA halpotypes influence livestock phenotypes, such as meat quality and lifetime daily gains in pigs, and reproductive efficiencies in pigs and cattle. Using stem cells models, we have been able to demonstrate that mtDNA haplotypes induce phenotypic changes through modulation of DNA methylation patterns that, in turn, affect chromosomal gene expression patterns. Using tumour cells, we have also demonstrated differences in the onset and progression of tumours based on mtDNA haplotype alone.
Sun X, Johnson J, St. John JC (In press) Global DNA methylation synergistically regulates the nuclear and mitochondrial genomes in glioblastoma cells. Nucleic Acids Research.
Kelly RD, Mahmud A, McKenzie M, Trounce IA, St. John JC (2012) Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A. Nucleic Acids Research 40:10124-10138.
Dickinson A, Yeung KY, Donoghue J, Baker MJ, Kelly RDW, McKenzie M, Johns TG, St. John JC (2013) The regulation of mitochondrial DNA copy number in Glioblastoma cells. Cell Death & Differentiation 20:1644-1653.
Kelly RDW, Rodda AE, Dickinson A, Mahmud A, Nefzger CM, Lee W, Forsythe JS, Polo JM, Trounce IA, McKenzie M, Nisbet DR, St. John JC (2013) Mitochondrial DNA haplotypes define gene expression patterns in pluripotent and differentiating embryonic stem cells. Stem Cells 31:703-716.
Lee W, Johnson J, Gough DJ, Donoghue J, Cagnone GLM, Vaghjiani V, Brown KA, Johns TG, St. John JC (2015) Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny. Cell Death & Disease 6(2):e1664.
Cagnone GLM, Tsai T-S, Makanji Y, Matthews P, Gould J, Bonkowski MS, Elgass KD, Wong ASA, Wu LE, McKenzie M, Sinclair DA, St. John JC (2016) Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency. Scientific Reports 6:23229.